U.S. patent application number 10/499890 was filed with the patent office on 2006-09-21 for compositions comprising immunoreactive reagents and saponins, and methods of use thereof.
This patent application is currently assigned to Antigenics, Inc.. Invention is credited to CharlotteA Kensil.
Application Number | 20060210555 10/499890 |
Document ID | / |
Family ID | 23345378 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060210555 |
Kind Code |
A1 |
Kensil; CharlotteA |
September 21, 2006 |
Compositions comprising immunoreactive reagents and saponins, and
methods of use thereof
Abstract
The present invention relates to pharmaceutical compositions
that are useful for the prevention and treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
disease wherein the treatment of such disease would be improved by
an enhanced immune response, and methods of formulating the
compositions. The compositions comprise an immunoreactive reagent
(i.e., an antigen binding protein comprising an antigen binding
region and a region or regions of an antibody that mediate antibody
dependent immunological processes) and a saponin. The present
invention also relates to methods of using the compositions of the
invention for the prevention and/or treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
disease wherein the treatment of such disease would be improved by
an enhanced immune response.
Inventors: |
Kensil; CharlotteA;
(Milford, MA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Antigenics, Inc.
Lexington
MA
02421
|
Family ID: |
23345378 |
Appl. No.: |
10/499890 |
Filed: |
December 20, 2002 |
PCT Filed: |
December 20, 2002 |
PCT NO: |
PCT/US02/40910 |
371 Date: |
April 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60343265 |
Dec 21, 2001 |
|
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|
Current U.S.
Class: |
424/133.1 ;
424/155.1; 514/26; 514/33 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 35/00 20180101; Y02A 50/30 20180101; A61P 31/00 20180101; A61K
31/704 20130101; Y02A 50/407 20180101; A61P 31/12 20180101; A61P
37/04 20180101; A61K 39/395 20130101; A61K 2039/55577 20130101;
A61P 31/10 20180101; Y02A 50/386 20180101; Y02A 50/412 20180101;
A61K 39/39558 20130101; A61P 31/04 20180101; Y02A 50/403 20180101;
A61P 25/00 20180101; A61K 45/06 20130101; Y02A 50/41 20180101; A61P
33/00 20180101; Y02A 50/466 20180101; A61K 39/395 20130101; A61K
2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101; A61K
39/39558 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/155.1; 514/026; 514/033 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/704 20060101 A61K031/704 |
Claims
1. A pharmaceutical composition comprising a saponin and an
immunoreactive reagent, wherein said immunoreactive reagent
specifically binds an antigen selected from the group consisting of
a tumor-associated antigen, an antigen of an agent of infectious
disease, an antigen associated with a neurodegenerative disease,
and an antigen associated with an amyloid disease.
2. The pharmaceutical composition of claim 1, wherein the
immunoreactive reagent is an antibody.
3. The pharmaceutical composition of claim 2, wherein the amount of
saponin is at least 1 microgram.
4. The pharmaceutical composition of claim 3, wherein the amount of
saponin is 10 to 20 micrograms.
5. The pharmaceutical composition of claim 3, wherein the amount of
saponin is 20 to 100 micrograms.
6. The pharmaceutical composition of claim 3, wherein the amount of
saponin is 100 to 500 micrograms.
7. The pharmaceutical composition of claim 1, wherein the saponin
is QS-7, QS-17, QS-18, QS-21, QS-21-V1, or QS-21-V2.
8. The pharmaceutical composition of claim 7, wherein the saponin
is QS-21.
9. The pharmaceutical composition of claim 1, wherein the
immunoreactive reagent specifically binds a tumor-associated
antigen.
10. The pharmaceutical composition of claim 9, wherein the tumor is
a non-Hodgkin's lymphoma, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma, leukemia, polycythemia vera, lymphoma, multiple
myeloma, Waldenstrom's macroglobulinemia, or heavy chain disease
(B-cell lymphoma).
11. The pharmaceutical composition of claim 9, wherein the
tumor-associated antigen is Melan A, MART-1, MAGE-1, MAGE-3, BAGE,
GAGE-1, GAGE-2, tyrosinase, gp100, gp75, HER-2/neu, c-erb-B2, CEA,
PSA, MUC-1, CA-125, Stn, TAG-72, KSA (17-1A), PSMA, p53, RAS,
EGF-R, VEGF, GD2, GM2, GD3, Anti-Id, CD20, CD19, CD22, CD36,
Aberrant class II, B1, CD25, or BPV.
12. The pharmaceutical composition of claim 1, wherein the
immunoreactive reagent binds specifically to an antigen of an agent
of infectious disease.
13. The pharmaceutical composition of claim 12, wherein the
infectious disease is a viral disease.
14. The pharmaceutical composition of claim 13, wherein the viral
disease is viral meningitis, encephalitis, dengue or smallpox, or
is a viral disease caused by hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I (HSV-I), herpes simplex type U (HSV-II), rinderpest,
rhinovirus, echovirus, rotavirus, respiratory syncytial virus,
papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, small pox, human immunodeficiency virus
type I (HIV-I), human immunodeficiency virus type II (HIV-II), and
Epstein Barr virus.
15. The pharmaceutical composition of claim 1, wherein the
infectious disease is a bacterial disease.
16. The pharmaceutical composition of claim 15, wherein the
bacterial disease is caused by infection by mycobacteria
rickettsia, mycoplasma, neisseria, streptococcus, staphylococcus,
mycobacterium, tetanus, pertissus, anthrax, cholera, plague,
diptheria, or chlamydia.
17. The pharmaceutical composition of claim 1, wherein the
infectious disease is a protozoal disease.
18. The pharmaceutical composition of claim 17, wherein the
protozoa is leishmania, kokzidioa, trypanosoma, or malaria.
19. The pharmaceutical composition of claim 1, wherein the
immunoreactive reagent binds specifically to an antigen associated
with a neurodegenerative or amyloid disease.
20. The pharmaceutical composition of claim 19, wherein the
neurodegenerative or amyloid disease is Alzheimer's Disease,
age-related loss of cognitive function, senile dementia,
Parkinson's disease, amyotrophic lateral sclerosis, Wilson's
Disease, cerebral palsy, progressive supranuclear palsy, Guam
disease, Lewy body dementia, prion diseases, spongiform
encephalopathies, Creutzfeldt-Jakob disease, polyglutamine
diseases, Huntington's disease, myotonic dystrophy, Freidrich's
ataxia, ataxia, Gilles de la Tourette's syndrome, seizure
disorders, epilepsy, chronic seizure disorder, stroke, brain
trauma, spinal cord trauma, AIDS dementia, alcoholism, autism,
retinal ischemia, glaucoma, autonomic function disorder,
hypertension, neuropsychiatric disorder, schizophrenia, or
schizoaffective disorder, type II diabetes, amyloidoses associated
with chronic inflammatory disease, amyloidoses associated with
infectious disease, or myeloma.
21. The pharmaceutical composition of claim 20, wherein the antigen
is .beta.-amyloid, an oligomeric A.beta. complex, an ApoE4-A.beta.
complex, tau protein, a mutant amyloid precursor, a mutant of
presenillin, .alpha.-synuclein, a prion protein, or an antigenic
fragment of any of the foregoing proteins.
22. A method of treating or preventing cancer in an individual in
whom such treatment or prevention is desired, comprising
administering to the individual an amount of an immunoreactive
reagent that specifically binds to an antigen of said type of
cancer, and a saponin, which amount is effective to treat or
prevent cancer in the individual.
23. A method of treating or preventing an infectious disease in an
individual in whom such treatment or prevention is desired,
comprising administering to the individual an amount of an
immunoreactive reagent that specifically binds to an antigen of
said type of cancer, and a saponin, which amount is effective to
treat or prevent the infectious disease in the individual.
24. A method of treating or preventing a neurodegenerative or
amyloid disease in an individual in whom such treatment or
prevention is desired, comprising administering to the individual
an immunoreactive reagent that specifically binds to an antigen of
said neurodegenerative or amyloid disease, and a saponin, which
amount is effective to treat or prevent the neurodegenerative or
amyloid disease in the individual.
25. The method of claim 22, 23 or 24, wherein the immunoreactive
reagent is an antibody.
26. The method of claim 22, 23 or 24, wherein the amount of saponin
is at least 1 microgram.
27. The method of claim 26, wherein the amount of saponin is 10 to
20 micrograms.
28. The method of claim 27, wherein the amount of saponin is 20 to
100 micrograms.
29. The method of claim 27, wherein the amount of saponin is 100 to
500 micrograms.
30. The method of claim 22, 23 or 24, wherein the saponin is QS-7,
QS-17, QS-18, QS-21, QS-21-V1, or QS-21-V2.
31. The method of claim 30, wherein the saponin is QS-21.
32. A method of enhancing antibody dependent cellular cytotoxicity
or phagocytosis of a target in an individual in whom such
enhancement is desired, comprising administering to the individual
an amount of an immunoreactive reagent that specifically binds to
an antigen of said target, and a saponin, which amount is effective
to enhance antibody dependent cellular cytotoxicity or phagocytosis
of the target in the individual.
33. The method of claim 32, wherein the immunoreactive reagent is
an antibody.
34. The method of claim 32, wherein the amount of saponin is at
least 1 microgram.
35. The method of claim 34, wherein the amount of saponin is 10 to
20 micrograms.
36. The method of claim 34, wherein the amount of saponin is 20 to
100 micrograms.
37. The method of claim 34, wherein the amount of saponin is 100 to
500 micrograms.
38. The method of claim 32, wherein the saponin is QS-7, QS-17,
QS-18, QS-21, QS-21-V1, or QS-21-V2.
39. The method of claim 38, wherein the saponin is QS-21.
40. A method of enhancing passive immunotherapy in an individual in
whom such enhancement is desired, wherein said immunotherapy
comprises administration of an amount of an immunoreactive reagent
that specifically binds to an antigen of a target, said method
comprising further administering an amount of a saponin, which
amount is effective to enhance the passive immunotherapy in the
individual.
41. The method of claim 40, wherein the immunoreactive reagent is
an antibody.
42. The method of claim 40, wherein the amount of saponin is at
least 1 microgram.
43. The method of claim 42, wherein the amount of saponin is 10 to
20 micrograms.
44. The method of claim 42, wherein the amount of saponin is 20 to
100 micrograms.
45. The method of claim 42, wherein the amount of saponin is 100 to
500 micrograms.
46. The method of claim 40, wherein the saponin is QS-7, QS-17,
QS-18, QS-21, QS-21-V1, or QS-21-V2.
47. The method of claim 46, wherein the saponin is QS-21.
48. A kit comprising, in one or more containers, a saponin and an
immunoreactive reagent, wherein said immunoreactive reagent
specifically binds an antigen selected from the group consisting of
a tumor-associated antigen, an antigen of an agent of infectious
disease, an antigen associated with a neurodegenerative disease,
and an antigen associated with an amyloid disease.
49. The kit of claim 48, wherein the saponin and immunoreactive
reagent are in separate containers.
50. The kit of claim 49, wherein the immunoreactive reagent is
formulated for intravenous administration, and the saponin is
formulated for administration other than intravenous
administration.
51. The kit of claim 49, wherein the immunoreactive reagent is an
antibody.
52. The kit of claim 49, wherein the saponin is QS-21.
53. The kit of claim 49, wherein the immunoreactive reagent is an
antibody and the saponin is QS-21.
54. The method of claim 22, 23, 24, 32, or 40, wherein the
immunoreactive reagent and the saponin are administered at
different times from one another.
55. The method of claim 54, wherein the saponin is administered
prior to the immunoreactive reagent.
56. The method of claim 55, wherein the saponin is administered at
least one day prior to the immunoreactive reagent.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to pharmaceutical compositions
that are useful for the prevention and treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
disease wherein the treatment of such disease would be improved by
an enhanced immune response, and methods of formulating the
compositions. The compositions comprise an immunoreactive reagent
(i.e., an antigen binding protein comprising an antigen binding
region and a region or regions of an antibody that mediate antibody
dependent immunological processes) and a saponin. Such antibody
dependent processes include, but are not limited to, antibody
dependent cellular cytotoxicity and phagocytosis. The present
invention also relates to methods of using the compositions of the
invention for the treatment of infectious diseases, primary and
metastatic neoplastic diseases (i.e., cancer), neurodegenerative or
amyloid diseases, or any other disease wherein the treatment of
such disease would be improved by an enhanced immune response. Such
methods include passive immunotherapy (i.e., passive immunization
with an immunoreactive reagent, such as an antibody). Any such
passive immunotherapy may be enhanced by the co-administration of a
saponin, preferably QS-21.
2. BACKGROUND OF THE INVENTION
[0002] 2.1. Passive Immunotherapy
[0003] Passive immunotherapy (also termed passive immunization)
refers to the administration of an immunoreactive reagent (i.e., an
antibody) comprising, for example, an antigen binding region
directed against an epitope on a pathogen, tumor or pathogenic
protein, and an Fc receptor-binding region, directly to a patient.
The immunoreactive reagent can be given prophylactically to, for
example, inhibit infection, or therapeutically to reduce or
eliminate infection, to reduce or eliminate cancer cells, or to
clear or remove pathogenic proteins, e.g., protein aggregates or
deposits, as occurs in neurodegenerative and/or amyloidogenic
disease. This is distinguished from immunization of a patient with
a protein to induce an in vivo immune response to produce
antibodies. Such administration preferably results in the
stimulation of effector cells with Fc receptors capable of
interacting with the Fc portion (i.e., the Fc receptor binding
region) of the antibody or immunoreactive agent, resulting in
cellular immune functions such as antibody-dependent cellular
cytotoxicity (e.g., ADCC) or antibody-mediated opsonization and/or
phagocytosis directed against the cell, pathogen, or protein
possessing the epitope recognized by the immunoreactive agent. The
saponin-mediated enhancement of passive immunotherapy can occur
through stimulation of effector cells, i.e., induction and/or
activiation of the Fc receptors on such cells. The efficacy of
antibody-mediated tumor therapy which depends on FcR effector cell
functions can be modified by the use of specific cytokines. Keler,
et al., 2000, J. Immunol. 164:5746-5752.
[0004] 2.2. Saponins
[0005] Quillaja saponins are a mixture of triterpene glycosides
extracted from the bark of the tree Quillaja saponaria. They have
long been recognized as immune stimulators that can be used as
vaccine adjuvants, (Campbell and Peerbaye, 1992, Res. Immunol.
143(5):526-530), and a number of commercially available complex
saponin extracts have been utilized as adjuvants. Crude saponins
have been extensively employed as adjuvants in veterinary vaccines
against foot and mouth disease, and in amplifying the protective
immunity conferred by experimental vaccines against protozoal
parasites such as malaria, Trypanosoma cruzi plasmodium, and the
humoral response to sheep red blood cells (SRBC) (Bomford, 1982,
Int. Arch. Allerg Appl. Immun. 67:127).
[0006] The first commercially available Quillaja saponin adjuvants
were crude extracts which, because of their variability, were not
desirable for use in veterinary practice or in pharmaceutical
compositions for man. An early attempt to purify Quillaja saponin
adjuvants was made by Dalsgaard (1974, Archiv fuer die gesamte
Virusforschung 44:243). Dalsgaard partially purified an aqueous
extract of the saponin adjuvant material from Quillaja saponaria
Molina. However, while Dalsgaard's preparation, "Quil-A," was a
definite improvement over the previously available commercial
saponins, it still exhibited considerable heterogeneity.
[0007] Subsequent analysis via high-pressure liquid chromatography
showed that Quil A was in fact a heterogeneous mixture of
structurally related triterpene glycosides (U.S. Pat. No.
5,057,540; Kersten et al., 1988, Infect. Immun. 56:432-438; Kensil
et al., 1991, J. Immunol. 146:431-437; Kensil et al., 1991, J. Am.
Vet. Med. Assoc. 199:1423-1427). However, not all of these saponins
were active as adjuvants.
[0008] The four most predominant purified Quillaja saponins are
QS-7, QS-17, QS-18, and QS-21 (alternatively identified as QA-7,
QA-17, QA-18, and QA-21). These saponins have been purified by HPLC
and low pressure silica chromatography and were found to be
adjuvant active, although differing in biological activities such
as hemolysis and toxicity in mice. In particular, QS-21 and QS-7
were found to be least toxic in mice (Kensil et al., 1991, J.
Immunol. 146:431-437).
[0009] Due to its potent adjuvant activity and low toxicity, QS-21
(commercially available as the "Stimulon.RTM." adjuvant) has been
identified as a useful immunological adjuvant (Kensil et al., 1995,
"Structural and Immunological Characterization of the Vaccine
Adjuvant QS-21," in Vaccine Design: The Subunit and Adjuvant
Approach, Powell and Newman eds., Plenum Press, New York). QS-21 is
a complex triterpene glycoside of quillaic acid. QS-21 is
glycosylated at triterpene carbon 3, triterpene carbon 28, and
carbon 5 of the second fatty acyl unit in a fatty acid domain.
[0010] More recently, QS-21 was further purified using hydrophilic
interaction chromatography (HILIC) and resolved into two peaks,
QS-21-V1 and QS-21-V2, which have been shown to be chemically
different compounds. In C57BL/6 mice immunized with vaccines
consisting of ovalbumin and either QS-21, QS-1-V1, or QS-21-V2,
both of the individual components QS-21-V1 and QS-21-V2 are
comparable in adjuvant effect to the original QS-21 peak
(containing a mixture of 3:2 QS-21-V1 and QS-21-V2) for boosting
the IgG subclasses IgG1, IgG2b, and IgG2 as well as the total IgG
titer (U.S. Pat. No. 5,583,112, the entire contents of which are
hereby incorporated by reference). Quillaja saponins are
structurally distinct from the saponins derived from other plant
species. Two structural features that distinguish Quillaja
saponaria saponins from those of other plant species are a fatty
acid domain and a triterpene aldehyde at carbon 4 of the
triterpene. (Kensil et al., 1995, "Structural and Immunological
Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design:
The Subunit and Adjuvant Approach, Powell and Newman eds., Plenum
Press, New York). Modifications to the aldehyde on the triterpene
indicate that this functional group may be involved in the adjuvant
mechanism (Soltysik et al., 1995, Vaccine 13(15):1403-1410).
[0011] Quillaja saponins, particularly QS-7, QS-17, QS-18, and
QS-21, have been found to be excellent stimulators of antibody
response to soluble T-dependent protein antigens, "subunit
antigens," which are poorly immunogenic and require a potent
adjuvant for maximization of immune responses. Examples of purified
subunit antigens for which saponin adjuvants that augment the IgG
response in mice include keyhole limpet hemocyanin (KLH), HIV-1
gp120 (Bomford et al., 1992, AIDS Res. Hum. Retroviruses 8:1765),
and influenza nucleoprotein (Brett et al., 1993, Immunology
80:306). QS-7, QS-17, QS-18, and QS-21 have also been shown to
stimulate potent antibody responses in mice to the antigens bovine
serum albumin and cytochrome b.sub.5 (Kensil et al., 1991, J.
Immunol. 146:431). The level of antibody response induced by these
purified saponins was comparable to other commonly used adjuvants,
e.g., complete Freund's adjuvant, and superior to aluminum
hydroxide.
[0012] QS-21 has also been shown to enhance antibody responses to
T-independent antigens, including unconjugated bacterial
polysaccharides (White et al., 1991, "A purified saponin acts as an
adjuvant for a T-independent antigen," in: Immunobiology of
Proteins and Peptides, Vol. VI (Atassi ed.), Plenum Press, New
York, pp. 207-210). The immunogenicity of the vaccine was further
increased by conjugating diphtheria toxoid to the polysaccharide.
QS-21 enhanced the antibody response to the polysaccharide as well
as the carrier, including IgG2a, IgG2b, and IgG3 responses
(Coughlin et al., 1995, Vaccine 13(1):17-21).
[0013] The ability of adjuvants to modulate the isotype
distribution and IgG subclass distribution of antibody response to
an antigen through the promotion of Ig subclass switching has
important implications for immunity to many bacterial and viral
vaccines. QS-7, QS-17, QS-18, and QS-21 stimulate IgG2a response to
cytochrome b5 after administration with saponin doses of 20 .mu.g
(Kensil et al., 1991, J. Immunol 146:431). In this regard, QS-21
shifts predominant IgG1 responses to a profile that includes
significant IgG2b and IgG2a responses. For example, QS-21 has been
shown to stimulate antigen-specific IgG2a to a number of antigens,
including Borrelia burgdorferi outer surface proteins OspA and OspB
(Ma et al., 1994, Vaccine 12(10):925), feline leukemia virus (FeLV)
envelope gp70 (Kensil et al., 1991, J. Am. Vet. Med. Assoc.
10:1423), human cytomegalovirus (HCMV) envelope protein gB (Britt
et al., 1995, J. Infect. Dis. 171:18), respiratory synctial virus
(RSV) purified fusion protein (Hancock et al., 1995, Vaccine
13(4):391), and tetanus toxoid (Coughlin et al., 1995, Vaccine
13(1):17). QS-21 has also been shown to induce boostable antibody
responses (Britt et al., 1995, J. Infect. Dis. 171:18-25; Helling
et al., 1995, Cancer Res. 55:2783-2788).
[0014] The ability of the QS-21 adjuvant to induce class I major
histocompatibility complex (MC) antigen-restricted cytotoxic
T-lymphocyte responses (CTL) after immunization with soluble
proteins is a characteristic of saponin adjuvants. A number of
studies have shown the ability of QS-21 to induce potent cytotoxic
T-lymphocyte (CTL) responses to various antigens, including
ovalbumin (Wu et al., 1994, Cell. Immunol. 154:394-406; Newman et
al., 1992, J. Immunol 148(8):2357-2362), recombinant HIV-1 gp160
protein (Wu et al., 1992, J. Immunol. 148:1519), respiratory
syncytial virus ("RSV") purified fusion protein (Hancock et al.,
1995, Vaccine 13(4):391), and subunit SIV.sub.mac251 gag and env
(Newman et al., 1994, AIDS Res. Hum. Retroviruses 10(7):853).
[0015] Most of the saponin adjuvant studies have been carried out
in mice. However, the adjuvant activity of saponins is not limited
to mice; it has also been demonstrated in humans, cats, dogs,
guinea pigs, rabbits, pigs, sheep, cattle, and nonhuman primates.
(Kensil et al., 1995, "Structural and Immunological
Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design:
The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J.
eds., Plenuim Press, New York).
[0016] Phase 1 human trials of QS-21 with GM2 ganglioside-keyhole
limpet haemocyanin conjugate vaccine have been conducted in
patients with malignant melanoma (Livingston et al., 1994, Vaccine
12:1275-1280. Increased immunogenicity after administration with
QS-21 adjuvant was observed (Helling et al., 1995, Cancer Res.
55:2783-2788). In another set of clinical trials, QS-21 was found
to be a potent immunological adjuvant that significantly increased
the serological response of melanoma patients to the murine
anti-idiotype antibody MELIMMUNE-1 (Livingston et al., 1995,
Vaccine Res. 4(2):87).
[0017] The immune adjuvant effect of saponins is dependent upon
dose. Depending upon the antigen and the species, a minimum dose
level of QS-21 is required for optimum response (Kensil et al.,
1991, J. Immunol. 146(2):431-7; Kensil et al., 1993, Ann NY Acad
Sci. 690:392-5; Newman et al., 1992, J. Immunol. 148(5):1519-25;
Livingston et al., 1994, Vaccine 12(14):1275-80). Below this
minimum dose, the immune adjuvant effect is suboptimal (either low
level or absent). QS-7 also has a dose response curve (Kensil et
al., 1991, J. Immunol. 146(2):431-7).
[0018] Saponins have also been discovered to elicit an innate
immune response which is independent of any particular antigen. The
innate immunity stimulated by saponins results in a potentiated
immune system that is capable of responding to an immunological
challenge in an enhanced manner. For example, saponins are capable
of increasing the production of TNF-alpha, IL-6 and MIP-1-alpha in
macrophage cells. In bone marrow derived dendritic cells, saponins
increase the production of MIP-1-alpha and IL-1, decrease the
production of E-12 and MIP-1-beta. This effect of saponins is
described in International Patent Publication-No. WO 01/51083,
incorporated herein in its entirety. This property of saponins is
different from their adjuvant effects in that an adjuvant effect is
specific to the particular antigen with which the adjuvant is
administered, while the innate immunity stimulation effect results
in a general enhancement of the immune system and its ability to
respond to a challenge which is independent of the particular
antigen used to challenge. Measurements of innate immunity, and
methods of determining enhancement thereof are known in the art,
and are described in International Patent Publication No. WO
01/51083.
3. SUMMARY OF THE INVENTION
[0019] The present invention relates to pharmaceutical compositions
that are useful for the prevention and treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
disease wherein the treatment of such disease would be improved by
an enhanced immune response, and methods of formulating the
compositions. The compositions comprise an immunoreactive reagent
(i.e., an antigen binding protein comprising an antigen binding
region and a region that mediates one or more antibody dependent
immunological processes) and a saponin. The compositions may
further comprise an immunostimulatory oligonucleotide.
[0020] The present invention also relates to methods of using the
compositions of the invention for the treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
treatment of a disease that would be improved by an enhanced immune
response. Such methods include passive immunotherapy. Any such
passive immumotherapy may be enhanced by the co-administration of a
saponin, preferably QS-21. The saponin may be administered
concurrently with the immunoreactive reagent, or within a short
time either before or after the administration of the
immunoreactive reagent. A short time includes, but is not limited
to 1, 15 or 30 minutes, 1, 6 or 12 hours, or 1 or 2 days. In a
preferred embodiment, the saponin is QS-7, QS-17, QS-18, QS-21,
QS-21-V1, or QS-21-V2.
[0021] The compositions of the invention can be used to generate an
immune response against epitopes associated with neurodegenerative
or amyloid diseases, cancer or an agent of infectious disease or
any cell or molecule bearing an epitope associated with the
aforementioned diseases, by administering to an individual a
therapeutic amount of the immunoreactive reagent and saponin. Where
an immune response against a type of cancer is desired, an
immunoreactive reagent is used which specifically binds to (or
"recognizes") an antigen of the type of cancer, i.e., a
tumor-associated antigen. Where eliciting an immune response
against an agent of an infectious disease is desired, an
immunoreactive reagent is used which specifically binds to an
antigen or pathologic protein (i.e., toxin) of the agent of
infectious disease. In other embodiments, the compositions of the
invention that comprise an immunoreactive reagent that specifically
binds to an antigen of a type of cancer are used to treat or
prevent the type of cancer; and the compositions of the invention
that comprise an immunoreactive reagent that specifically binds to
an agent of an infectious disease are used to treat or prevent the
infectious disease. In other embodiments, the compositions of the
invention comprise an immunoreactive reagent that specifically
binds an antigenic molecule associated with a neurodegenerative
disease or an amyloid disease ane are used to treat or prevent said
neurodegenerative or amyloid disease.
[0022] Prophylactic and therapeutic dosage regimens and kits are
also provided by the invention.
4. DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention relates to pharmaceutical compositions
that are useful for the prevention and treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
disease wherein the treatment of such disease would be improved by
an enhanced immune response, and methods of formulating the
compositions. In certain embodiments, the outcome of eliciting an
immune response is prophylaxis or therapy. The compositions of the
invention can be used to provide improved passive immunotherapy
against cancer or an agent of infectious disease or
neurodegenerative/amyloid diseases or any other disease or
pathological state that can be treated by passive immunotherapy, by
administering to an individual a therapeutic amount of the
immunoreactive reagent and saponin.
[0024] Cancers that can be treated according to the methods of the
invention include, but are not limited to, leukemia (e.g., acute
leukemia such as acute lymphocytic leukemia and acute myelocytic
leukemia), neoplasms, tumors (e.g., non-Hodgkin's lymphoma,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma), heavy chain disease (B-cell lymphoma), metastases,
or any disease or disorder characterized by uncontrolled cell
growth. Tumor antigens or tumor associated antigens include
cancer-germ cell (CG) antigens (MAGE, NY-ESO-1), mutational
antigens (MUM-1, p53, CDK4), over-expressed self-antigens (p53,
HER2/NEU), viral antigens (from Papilloma Virus, Epstein-Barr
Virus), tumor proteins derived from non-primary open reading frame
mRNA sequences (NY-ESO1, LAGE1), Melan A, MART-1, MAGE-1, MAGE-3,
BAGE, GAGE-1, GAGE-2, tyrosinase, gp100, gp75, HER-2/neu, c-erb-B2,
CEA, PSA, MUC-1, CA-125, Stn, TAG-72, KSA (17-1A), PSMA, p53 (point
mutated and/or overexpressed), RAS (point mutated), EGF-R, VEGF,
GD2, GM2, GD3, Anti-Id, CD20, CD19, CD22, CD36, Aberrant class II,
B1, CD25 (IL-2R) (anti-TAC), or HPV.
[0025] Infectious agents that can be treated according to the
invention include, but are not limited to viruses, such as
hepatitis type A, hepatitis type B, hepatitis type C, influenza,
varicella, adenovirus, herpes simplex type I (HSV-I), herpes
simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, small pox, Epstein Barr virus, human immunodeficiency virus
type I (HIV-I), human immunodeficiency virus type II (HIV-II), and
agents of viral diseases such as viral miningitis, encephalitis,
dengue or small pox; bacteria, such as mycobacteria rickettsia,
mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi (Lyme
disease), Bacillus antracis (anthrax), tetanus, streptococcus,
staphylococcus, mycobacterium, tetanus, pertissus, cholera, plague,
diptheria, chlamydia, S. aureus and legionella; and agents of
protozoal disease, such as leishmania, kokzidioa, trypanosoma or
malaria.
[0026] Immunoreactive reagents specifically binding an antigenic
molecule in or on a cell or structure, e.g., extracellular deposits
or plaques comprising peptide and/or protein fibrils, that displays
the hallmarks of a neurodegenerative or amyloid disease may also be
utilized. Preferably, where it is desired to treat or prevent
neurodegenerative or amyloid diseases, immunoreactive reagents that
specifically bind to molecules comprising epitopes of antigenic
molecules associated with neurodegenerative diseases, or epitopes
of antigenic molecules associated with amyloid diseases, including
but not limited to fibril peptides or proteins, are used. Such
neurodegenerative disease-associated antigenic molecules may be
molecules associated with Alzheimer's Disease, age-related loss of
cognitive function, senile dementia, Parkinson's disease,
amyotrophic lateral sclerosis, Wilson's Disease, cerebral palsy,
progressive supranuclear palsy, Guam disease, Lewy body dementia,
prion diseases, spongiform encephalopathies, Creutzfeldt-Jakob
disease, polyglutamine diseases, Huntington's disease, myotonic
dystrophy, Freidrich's ataxia, ataxia, Gilles de la Tourette's
syndrome, seizure disorders, epilepsy, chronic seizure disorder,
stroke, brain trauma, spinal cord trauma, AIDS dementia,
alcoholism, autism, retinal ischemia, glaucoma, autonomic function
disorder, hypertension, neuropsychiatric disorder, schizophrenia,
or schizoaffective disorder. Examples of such antigentic molecules
are disclosed in U.S. application Ser. No. 09/489,216, which is
incorporated by reference herein in its entirety, and include, but
are not limited to, .beta.-amyloid or a fragment thereof, an
oligomeric A.beta. complex or a fragment thereof, an ApoE4-A.beta.
complex, tau protein or a fragment thereof, amyloid precursor
protein or a fragment thereof, a mutant amyloid precursor protein
or a fragment thereof, presenillin or a fragment thereof, a mutant
of presenillin or a fragment thereof, .alpha.-synuclein or a
fragment thereof, or a prion protein or a fragment thereof, and the
antigenic derivatives of any of the foregoing proteins or fragments
thereof. Amyloid disease associated antigenic molecules may be
molecules associated with diseases characterized by the
extracellular deposition of protein and/or peptide fibrils which
form amyloid deposits or plaques, including but not limited to type
U diabetes and amyloidoses associated with chronic inflammatory or
infectious disease states and malignant neoplasms, e.g., myeloma.
Certain amyloid disease such as Alzheimner's disease and prion
diseases, e.g., Creutzfeldt Jacob disease, are neurodegenerative
diseases.
[0027] The treatment of other disease or pathogenic states that can
be treated by immunotherapy may are also within the scope of the
present invention. Such treatment includes the treatment of
cardiovascular disease with an immunoreactive reagent that binds
angiotensin 2, and the treatment of autoimmune associated diseases,
such as arthritis, with immunoreactive reagents that bind IL-10,
tumor necrosis factor, or other immunoregulatory molecules.
[0028] The compositions comprise an immunoreactive reagent (i.e.,
an antigen binding protein comprising an antigen binding region and
a region that mediates one or more antibody dependent immunological
processes, e.g., an Fc receptor-binding region) and a saponin. In
certain embodiments, the compositions of the invention further
include an immunostimulatory oligonucleotide.
[0029] The present invention also relates to methods of using the
compositions of the invention for the treatment of infectious
diseases, primary and metastatic neoplastic diseases (i.e.,
cancer), neurodegenerative or amyloid diseases, or any other
treatment of a disease that would be improved by an enhanced immune
response. Such methods include passive immunotherapy. Any such
passive immunotherapy may be enhanced by the co-administration of a
saponin, preferably QS-21. The saponin may be administered
concurrently with the immunoreactive reagent, or within a short
time either before or after the administration of the
immunoreactive reagent. A short time includes, but is not limited
to 1, 15 or 30 minutes, 1, 6, 12, 18 or 36 hours, or 1 or 2
days.
[0030] In a preferred embodiment, the saponin is QS-7, QS-21,
QS-21-V1, or QS-21-V2. In another embodiment, the saponin is an
active derivative, e.g., semi-synthetic derivatives such as
GP1-100, as well as other immuostimulatory (e.g., adjuvant active)
saponins or saponin-containing compositions. The adjuvant,
haemolytic, and innate immune stimulatory activities of individual
saponins have been extensively studied in the art. (Lacaille-Dubois
and Wagner, 1996 "A review of the biological and pharmacological
activities of saponins." Phytomedicine vol. 2, pp 363-386,
incorporated herein by reference in its entirety). Such active
saponins include Quil A and fractions thereof, ISCOMS, saponins
derived from other plant species such as Gypsophila and Saponaria
(Bomford et al., 1992, Vaccine 10(9):572-577, incorporated herein
by reference in its entirety), and Chenopodium quinoa saponins have
been used in both intranasal and intragastric vaccines (Estrada et
al., 1998, Comp. Immunol. Microbiol. Infect. Dis. 21(3):225-36,
incorporated herein by reference in its entirety).
[0031] The compositions can be utilized for the prevention of a
variety of cancers, e.g., in individuals who are predisposed as a
result of familial history or in individuals with an enhanced risk
to cancer due to environmental factors, for the prevention of
infectious diseases, e.g., in individuals with enhanced risks of
exposure to agents of infectious disease, and for the prevention of
neurodegenerative or amyloid diseases, for example in individuals
with genetic predispositions to neurodegenerative or amyloid
diseases.
[0032] The invention also provides kits comprising one or more
containers with one or more of the ingredients of the
pharmaceutical compositions of the invention. Optionally associated
with such kit(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. In one embodiment, the kits can optionally further
comprise a predetermined amount of the immunoreative reagent (i.e.,
an antigen binding protein comprising an antigen binding region and
a region that mediates one or more antibody dependent immunological
processes, e.g., an Fc receptor-binding region) and a saponin. In a
preferred embodiment, the kit comprises the immunoreactive reagent
and the saponin in separate containers.
[0033] 4.1. Immunoreactive Reagents
[0034] The immunoreactive reagents of the invention are peptides
comprising 1) an antigen binding region and, optionally, 2) a
region that mediates one or more antibody dependent immunological
processes. The antigen binding region can comprise or consist of
the antigen binding region of an antibody. The antigen binding
region can comprise any peptide or domain that interacts
specifically with an antigen of interest. For example, the antigen
binding region can be a ligand or other specific binding partner of
the antigen of interest, or can be a fragment of such ligand or
binding partner, or can be derived from such ligand or binding
partner.
[0035] The region that mediates one or more antibody dependent
immunological processes can comprise or consist of a region that is
capable of binding an Fc receptor, e.g., the portion of an antibody
that binds Fc receptors, or a region that binds complement, e.g.,
the complement binding region of an antibody. This region can also
be an antigen binding domain of an antibody that binds to Fc
receptors or complement.
[0036] Immunoreactive reagents include antibodies, Fab and
F(ab').sub.2 fragments, molecules or proteins engineered to include
the antigen binding portion of an antibody, molecules or proteins
engineered to include an antigen binding domain that mediates
antibody dependent immune responses, a peptide or domain that
interacts specifically with the antigen of interest, or any antigen
binding domain that interacts with an antigen/epitope of interest,
and the domain of the constant region of an antibody that mediates
antibody dependent immune effector cell responses or processes.
Examples of such domains or regions within the Ab constant region
that can be used in the present invention include those disclosed
in Reddy et al., 2000, J. Immunol. 164(4):1925-33; Coloma et al.,
1997, Nat Biotechnol. 15(2):159-63; Carayannopoulos et al., 1994,
Proc Natl. Acad. Sci. U.S.A. 91(18):8348-52; Morrison, 1992, Annu
Recombinant expression vector Immunol. 10:239-65; Traunecker et
al., 1992, Int. J. Cancer Suppl., 7:51-2; Gillies et al., 1990,
Hum. Antibodies Hybridomas, 1 (1):47-54; each of which is
incorporated herein by reference in its entirety.
[0037] Such antibody dependent processes include, but are not
limited to, antibody dependent cellular cytotoxicity, activation of
complement, opsonization and phagocytosis. The effector cells that
mediate certain antibody dependent processes include monocytes,
macrophages, natural killer cells, and polymorphonuclear cells.
Without being bound by a particular mechanism, it is thought that
saponins are able to increase receptors on the effector cells
responsible for mediating the antibody dependent response. These
receptors include the Fc alpha and Fc gamma receptors, isoforms
thereof, or any combination thereof. Thus, in a particular
embodiment, the region of the immunoreactive reagent that mediates
one or more antibody dependent immunological processes comprises or
consists of a region that is a ligand for Fc receptors, preferably
the Fc a receptor or the Fc gamma receptor, or both. In another
embodiment, the region of the immunoreactive reagent that mediates
one or more antibody dependent immunological processes comprises or
consists of a region that stimulates the function of immune
effector cells, preferably monocytes, macrophages, natural killer
cells, polymorphonuclear cells, or any combination of two or more
of such cells, such that a prophylactic and/or therapeutic effect
is achieved.
[0038] In a preferred embodiment, the immunoreactive reagent is an
antibody, or a composition comprising an antibody such as serum. In
a particular embodiment, the immunoreactive reagent is an IgA, IgG
or IgM antibody, or comprises a fragment thereof. In a particularly
preferred embodiment, the immunoreactive reagent is a monoclonal
antibody, or includes fragments of a monoclonal antibody. The
immunoreactive reagent may also comprise or consist of human immune
globulin for treatment of Hepatitis B; Respigam for the treatment
of RSV; Sandoglobulin, or ImmuneGlobulin IV (IGIV). In another
embodiment, the immunoreactive reagent is not directed towards any
single epitope, but instead comprises a mixture of one or more
molecules that bind to a population of epitopes. An example of such
an immunoreactive reagent is serum or antibodies concentrated from
serum or plasma. Such serum or plasma may be from a subject
immunized against a particular antigen, or from a subject not so
immunized.
[0039] In another embodiment, the immunoreactive reagent is a
bi-specific molecule having two antigen binding regions of
different specificity, i.e., one recognizing an epitope on a target
cell or protein, and the other recognizing an epitope of an
effector cell, e.g., an epitope of FcR. In another embodiment, the
immunoreactive reagent is a bi-specific molecule having two antigen
binding domains for different epitopes on the target cell/protein
and a domain that mediates antibody dependent immune responses.
Such bi-specific molecules that target cancer cells or pathogens
and their therapeutic effects have been examined both in vivo and
in vitro (e.g., Wallace et al., 2001, J Immunol. Methods
248(1-2):167-82; Sundarapandiyan et al., 2001, J Immunol. Methods
248(1-2):113-23; Honeychurch et al., 2000, Blood 96(10):3544-52;
Negri et al., 1995, Br J Cancer 72(4):928-33; Wang et al., 1994,
Zhonghua Zhong Liu Za Zhi 16(2):83-7, Chinese) (each of which is
incorporated herein by reference in its entirety).
[0040] In a preferred embodiment, the immunoreactive reagent is
purified. "Purified" as used herein to describe certain peptides,
antibodies, molecules, proteins, antigens, saponins, and the like,
refer to a state beyond that in which the molecules, proteins,
antigens, and the like, are separated from greater than 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the
proteins, polysaccharides, and/or lipids with which the peptides,
antibodies, molecules, proteins, antigens, saponins, and the like
are normally associated naturally. If the isolated molecules,
proteins, antigens, saponins, and the like are synthesized, they
are contaminated with less than 50%, 40%, 30%, 20%, 10%, 5%, 1% or
0.1% of the chemical precursors or synthesis reagents used to
synthesize the molecules, proteins, antigens, saponins, and the
like. In preferred embodiments the peptides, antibodies, molecules,
proteins, antigens, saponins, and the like are at least 1% pure, 5%
pure, 10% pure, 20% pure, 30% pure, 40% pure, 50% pure, 60% pure,
70% pure, 80% pure, 90% pure, 95% pure, 99% pure, or 100% pure. As
used herein, the term "% pure" indicates the percentage of the
total composition that is made up of the molecule of interest, by
weight. Thus, a composition of 100 grams containing 50 grams of a
molecule of interest is 50% pure with respect to the molecule of
interest.
[0041] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681
(Elsevier, N.Y., 1981) (both of which are incorporated herein by
reference in their entireties). The term "monoclonal antibody" as
used herein is not limited to antibodies produced through hybridoma
technology. The term "monoclonal antibody" refers to an antibody
that is derived from a single clone, including any eukaryotic,
prokaryotic, or phage clone, and not the method by which it is
produced.
[0042] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with an antigen of
interest or a cell expressing such an antigen. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells. Hybridomas are selected
and cloned by limiting dilution. The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding the antigen. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
inoculating mice intraperitoneally with positive hybridoma
clones.
[0043] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the complete light chain, and the variable
region, the CHI region and the hinge region of the heavy chain.
[0044] For example, antibodies can also be generated using various
phage display methods known in the art. In phage display methods,
functional antibody domains are displayed on the surface of phage
particles which carry the polynucleotide sequences encoding them.
In a particular embodiment, such phage can be utilized to display
antigen binding domains, such as Fab and Fv or disulfide-bond
stabilized Fv, expressed from a repertoire or combinatorial
antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be
selected or identified with antigen, e.g., using labeled antigen or
antigen bound or captured to a solid surface or bead. Phage used in
these methods are typically filamentous phage, including fd and
M13. The antigen binding domains are expressed as a recombinantly
fused protein to either the phage gene III or gene VIII protein.
Examples of phage display methods that can be used to make the
immunoglobulins, or fragments thereof, of the present invention
include those disclosed in Brinkman et al., 1995, J. Immunol.
Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.,
24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,
1994, Advances in Immunology 57:191-280; PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0045] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired fragments, and expressed in any desired host,
including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as described in detail below. For example,
techniques to recombinantly produce Fab, Fab' and F(ab').sub.2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
1992, BioTechniques 12(6):864-869; and Sawai et al., 1995, AJRI
34:26-34; and Better et al., 1988, Science 240:1041-1043 (each of
which is incorporated by reference in its entirety). Examples of
techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88;
Shu et al., 1993, PNAS 90:7995-7999; and Skerra et al., 1988,
Science 240:1038-1040.
[0046] For some uses, including in vivo use of antibodies in
humans, it may be preferable to use chimeric, humanized, or human
antibodies. A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a constant region derived from a human
immunoglobulin. Methods for producing chimeric antibodies are known
in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and
4,816,397, which are incorporated herein by reference in their
entireties. Humanized antibodies are antibody molecules from
non-human species that bind the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Reichmann et al., 1988, Nature 332:323, which
are incorporated herein by reference in their entireties.
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et
al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994,
Proc Natl. Acad. Sci. USA, 91:969-973), and chain shuffling (U.S.
Pat. No. 5,565,332), all of which are hereby incorporated by
reference in their entireties.
[0047] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887
and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO
98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741,
each of which is incorporated herein by reference in its
entirety.
[0048] Human antibodies can also be produced-using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publications WO 98/24893;
WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598
877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and
5,939,598, which are incorporated by reference herein in their
entireties. In addition, companies such as Abgenix, Inc. (Freemont,
Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0049] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., 1988, Bio/technology 12:899-903).
[0050] In a preferred embodiment, the antibodies have in vivo
therapeutic and/or prophylactic uses. Examples of therapeutic and
prophylactic antibodies include, but are not limited to,
SYNAGIS.RTM. (MedImmune, MD) which is a humanized anti-respiratory
syncytial virus (RSV) monoclonal antibody for the treatment of
patients with RSV infection; HERCEPTIN.RTM. (Trastuzumab)
(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody
for the treatment of patients with metastatic breast cancer;
REMICADE.RTM. (infliximab) (Centocor, PA) which is a chimeric
anti-TNF.alpha. monoclonal antibody for the treatment of patients
with Crone's disease; REOPRO.RTM. (abciximab) (Centocor) which is
an anti-glycoprotein IIb/IIIa receptor on the platelets for the
prevention of clot formation; ZENAPAX.RTM. (daclizumab) (Roche
Pharmaceuticals, Switzerland) which is an immunosuppressive,
humanized anti-CD25 monoclonal antibody for the prevention of acute
renal allograft rejection. Other examples are a humanized anti-CD18
F(ab').sub.2 (Genentech); CDP860 which is a humanized anti-CD 18
F(ab').sub.2 (Celltech, UK); PRO542 which is an anti-HIV gp120
antibody fused with CD4 (Progenics/Genzyme Transgenics); Ostavir
which is a human anti Hepatitis B virus antibody (Protein Design
Lab/Novartis); PROTOVIR.TM. which is a humanized anti-CMV IgG1
antibody (Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a
murine anti-TNF-.alpha. F(ab').sub.2 (Knoll Pharma/BASF); IC14
which is an anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF
IgG1 antibody (Genentech); OVAREX.TM. which is a murine anti-CA 125
antibody (Altarex); PANOREX.TM. which is a murine anti-17-IA cell
surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2
which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone
System); IMC-C225 which is a chimeric anti-EGFR IgG antibody
(ImClone System); VITAXIN.TM. which is a humanized
anti-.alpha.V.beta.3 integrin antibody (Applied Molecular
Evolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti
CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized
anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITXANT which
is a chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech,
Roche/Zettyaku); LYMPHOCIDE.TM. which is a humanized anti-CD22 IgG
antibody (Immunomedics); Smart ID10 which is a humanized anti-HLA
antibody (Protein Design Lab); ONCOLYM1M (Lym-1) is a radiolabelled
murine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone); ABX-IL8
is a human anti-IL8 antibody (Abgenix); anti-CD11a is a humanized
IgG1 antibody (Genentech/Xoma); ICM3 is a humanized anti-ICAM3
antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody
(IDEC Pharm/Mitsubishi); ZEVALIN.TM. is a radiolabelled murine
anti-CD20 antibody (DEC/Schering AG); IDEC-131 is a humanized
anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4
antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody
(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG
(Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5
(C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-.alpha.
antibody (CAT/BASF); CDP870 is a humanized anti-TNF-.alpha. Fab
fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1
antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human
anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized
anti-TNF-.alpha. IgG4 antibody (Celltech); LDP-02 is a humanized
anti-.alpha.4.beta.7 antibody (LeukoSite/Genentech); OrthoClone
OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech);
ANTOVA.TM. is a humanized anti-CD40L IgG antibody (Biogen);
ANTEGREN.TM. is a humanized anti-VLA-4 IgG antibody (Elan); MDX-33
is a human anti-CD64 (Fc.gamma.R) antibody (Medarex/Centeon);
SCH55700 is a humanized anti-IL-5 IgG4 antibody
(Celltech/Schering); SB-240563 and SB-240683 are humanized
anti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham);
rhuMab-E25 is a humanized anti-IgE IgG1 antibody
(Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a murine anti
CD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG
antibody (Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine
anti-CD3 IgG2a antibody (ortho Biotech); SIMULEC.TM. is a chimeric
anti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanized
anti-.beta..sub.2-integrin IgG antibody (LeukoSite); Anti-LFA-1 is
a murine anti CD18 F(ab').sub.2 (Pasteur-Merieux/Immunotech);
CAT-152 is a human anti-TGF-.beta..sub.2 antibody (Cambridge Ab
Tech); and Corsevin M is a chimeric anti-Factor VII antibody
(Centocor). The above-listed immunoreactive reagents, as well as
any other immunoreactive reagents, may be administered according to
any regimen known to those of skill in the art, including the
regimens recommended by the suppliers of the immunoreactive
reagents. Immunoreactive reagents that bind to 4-1BB glycoprotein
or Cytotoxic T Lymphocyte Antigen-4 (CTLA-4), such as antibodies
specific for 4-1BB or CTLA-4, respectively, are also contemplated
within the present invention.
[0051] The immunoreactive reagents of the invention can be produced
by any method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques. Such methods are described below with
reference to an antibody immunoreactive reagent, but are readily
applicable to the production of other immunoreactive reagents.
[0052] The nucleotide sequence encoding an antibody or other
immunoreactive reagent may be obtained from any information
available to those of skill in the art (i.e., from Genbank, the
literature, or by routine cloning). If a clone containing a nucleic
acid encoding a particular antibody or an epitope-binding fragment
thereof or other immunoreactive reagent is not available, but the
sequence of the antibody molecule or epitope-binding fragment
thereof or other immunoreactive reagent is known, a nucleic acid
encoding the immunoglobulin or other immunoreactive reagent may be
chemically synthesized or obtained from a suitable source (e.g., an
antibody cDNA library, or a cDNA library generated from, or nucleic
acid, preferably poly A+ RNA, isolated from any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR may
then be cloned into replicable cloning vectors using any method
well known in the art. In the case of immunoreactive reagents that
do not exist in nature, nucleic acids encoding the different
regions of the immunoreactive reagent can be obtained from
preexisting libraries or known genes, or can be synthesized.
[0053] Once the nucleotide sequence of the antibody or other
immunoreactive reagent is determined, the nucleotide sequence of
the antibody or other immunoreactive reagent may be manipulated
using methods well known in the art for the manipulation of
nucleotide sequences, e.g., recombinant DNA techniques, site
directed mutagenesis, PCR, etc. (see, for example, the techniques
described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.; and Ausubel et al., eds., 1998, Current Protocols in
Molecular Biology, John Wiley & Sons, NY, which are both
incorporated by reference herein in their entireties), to generate
antibodies or other immunoreactive reagent having a different amino
acid sequence by, for example, introducing amino acid
substitutions, deletions, and/or insertions into the
epitope-binding domain regions of the antibodies or other
immunoreactive reagent or into the constant (Fc) regions of the
antibodies or other immunoreactive reagent which are involved in
the interaction with immune effector cells.
[0054] Recombinant expression of an antibody or other
immunoreactive reagent requires construction of an expression
vector containing a nucleotide sequence that encodes the antibody
or other immunoreactive reagent. Once a nucleotide sequence
encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably, but not necessarily,
containing the heavy or light chain variable region) or other
immunoreactive reagent has been obtained, the vector for the
production of the antibody molecule or other immunoreactive reagent
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody or other
immunoreactive reagent encoding nucleotide sequence are described
herein. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing antibody or
other immunoreactive reagent coding sequences and appropriate
transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques, and id vivo genetic recombination. The
nucleotide sequence encoding the heavy-chain variable or constant
region, light-chain variable or constant region, both the
heavy-chain and light-chain variable regions, an epitope-binding
fragment of the heavy- and/or light-chain variable region, or one
or more complementarity determining regions (CDRs) of an antibody
or other immunoreactive reagent may be cloned into such a vector
for expression. The expression vector is transferred to a host cell
by conventional techniques and the transfected cells are then
cultured by conventional techniques.
[0055] A variety of host-expression vector systems may be utilized
to express the antibody molecules or other immunoreactive reagent
of the invention. Such host-expression systems represent vehicles
by which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody molecule or other immunoreactive
reagent of the invention in situ. These include, but are not
limited to, microorganisms such as bacteria (e.g., E. coli and B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody or other
immunoreactive reagent coding sequences; yeast (e.g., Saccharomyces
and Pichia) transformed with recombinant yeast expression vectors
containing antibody or other immunoreactive reagent coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody or other
immunoreactive reagent coding sequences; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; and tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing antibody or other immunoreactive reagent coding
sequences; and mammalian cell systems (e.g., COS, CHO, BHK, 293,
3T3 and NSO cells) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule or other immunoreactive
reagent, are used for the expression of a recombinant antibody or
other immunoreactive reagent molecule. For example, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., 1986, Gene 45:101, and Cockett et al.,
1990, Bio/Technology 8:2).
[0056] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule or other immunoreactive reagent being expressed.
For example, when a large quantity of such a protein is to be
produced, for the generation of pharmaceutical compositions of an
antibody molecule, vectors which direct the expression of high
levels of fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited to, the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791),
in which the antibody or other immunoreactive reagent coding
sequence may be ligated individually into the vector in frame with
the lacZ coding region so that a fusion protein is produced; and
pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109, and Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509).
[0057] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
or other immunoreactive reagent coding sequence may be cloned
individually into non-essential regions (for example the
polylhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
[0058] In mammalian host cells, a number of viral-based expression
systems may be utilized to express an antibody molecule or other
immunoreactive reagent of the invention. In cases where an
adenovirus is used as an expression vector, the antibody or other
immunoreactive reagent coding sequence of interest may be ligated
to an adenovirus transcription/translation control complex, e.g.,
the late promoter and tripartite leader sequence. This chimeric
gene may then be inserted in the adenovirus genome by in vitro or
in vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the antibody
molecule or other immunoreactive reagent in infected hosts (e.g.,
see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA
81:355-359). Specific initiation signals may also be required for
efficient translation of inserted antibody or other immunoreactive
reagent coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bitter et al., 1987, Methods in
Enzymol. 153:516-544).
[0059] In addition, a host cell strain may be chosen which
modulates the expression of the antibody or other immunoreactive
reagent sequences, or modifies and processes the antibody or other
immunoreactive reagent in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g. cleavage)
of protein products may be important for the function of the
antibody or other immunoreactive reagent. Different host cells have
characteristic and specific mechanisms for the post-translational
processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the
correct modification and processing of the antibody or other
immunoreactive reagent expressed. To this end, eukaryotic host
cells which possess the cellular machinery for proper processing of
the primary transcript, glycosylation, and phosphorylation of the
gene product may be used. Such mammalian host cells include but are
not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and
in particular, myeloma cells such as NSO cells, and related cell
lines, see, for example, Morrison et al., U.S. Pat. No. 5,807,715,
which is hereby incorporated by reference in its entirety.
[0060] For long-term, high-yield production of recombinant
antibodies or other immunoreactive reagent, stable expression is
preferred. For example, cell lines which stably express the
antibody molecule or other immunoreactive reagents may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the antibody molecule or other immunoreactive
reagent. Such engineered cell lines may be particularly useful in
screening and evaluation of compositions that interact directly or
indirectly with the antibody molecule or other immunoreactive
reagent.
[0061] A number of selection systems may be used, including but not
limited to, the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:8-17) genes can be employed in
tk.sup.-, hgprt.sup.- or aprt.sup.- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., 1980, Natl. Acad. Sci. USA 77:357, and O'Hare et
al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.
Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,
Ann. Rev. Biochem. 62: 191-217; and May, 1993, TIB TECH 11(5):155-2
15); and hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30:147). Methods commonly known in the art of
recombinant DNA technology may be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY;
in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current
Protocols in Human Genetics, John Wiley & Sons, NY; and
Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are
incorporated by reference herein in their entireties.
[0062] The expression levels of an antibody molecule or other
immunoreactive reagent can be increased by vector amplification
(for a review, see Bebbington and Hentschel, 1987, The use of
vectors based on gene amplification for the expression of cloned
genes in mammalian cells in DNA cloning, Vol. 3. Academic Press,
New York). When a marker in the vector system expressing antibody
or other immunoreactive reagent is 1 amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody or other immunoreactive reagent
gene, production of the antibody or other immunoreactive reagent
will also increase (Crouse et al., 1983, Mol. Cell. Biol.
3:257).
[0063] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides or different selectable markers to ensure
maintenance of both plasmids. Alternatively, a single vector may be
used which encodes, and is capable of expressing, both heavy and
light chain polypeptides. In such situations, the light chain
should be placed before the heavy chain to avoid an excess of toxic
free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980,
Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for the
heavy and light chains may comprise cDNA or genomic DNA.
[0064] Once an antibody or other immunoreactive reagent molecule of
the invention has been produced by recombinant expression, it may
be purified by any method known in the art for purification of an
immunoglobulin molecule or other immunoreactive reagent, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A
purification, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard techniques for
the purification of proteins. Further, the antibodies or other
immunoreactive reagents of the present invention or fragments
thereof may be fused to heterologous polypeptide sequences
described herein or otherwise known in the art to facilitate
purification.
[0065] 4.2. Sources of Saponins
[0066] Any saponin or saponin preparation known in the art may be
used in the compositions and methods of the invention. The term
"saponin" as used herein includes glycosidic triterpenoid compounds
which produce foam in aqueous solution and have hemolytic activity
in most cases. The invention encompasses the use of saponins per
se, as well as natural and pharmaceutically acceptable salts and
pharmaceutically acceptable derivatives thereof. The term "saponin"
also embodies biologically active fragments thereof. The term
"saponin" also encompasses chemically modified saponins, such as
GPI-0100, and other modified saponins described, for example, in
U.S. Pat. Nos. 6,080,725; 5,977,081, and 5,443,829, each of which
is incorporated herein in its entirety. The use of modified
saponins adapted for, e.g., drug delivery, such as those described
in U.S. Pat. Nos. 5,650,398; 5,443,829 and 5,273,965, each of which
is incorporated herein in its entirety, is also encompassed by the
invention. Preferably, the saponin is a single saponin. In other
embodiments of the invention, the term "saponin" covers mixtures of
saponins. Suitable saponins include QS-7, QS-17, QS-18 and QS-21.
Preferably, the mixture of saponins comprises two or more
substantially pure saponins. More preferably, the two or more
substantially pure saponins are from Quillaja saponaria in doses
that are otherwise suboptimal for the individual saponins. In a
particularly preferred embodiment, the combination of saponins
consists essentially of two substantially pure saponins QS-7 and
QS-21 or, in other particularly preferred embodiments, QS-7 and
QS-21-V1 or QS-7 and QS-21-V2, as described in U.S. Pat. No.
6,231,859, which is herein incorporated by reference in its
entirety. As used herein, "substantially pure" means substantially
free from compounds normally associated with the saponin in its
natural state and exhibiting constant and reproducible
chromatographic response, elution profiles, and biologic activity.
The term "substantially pure" is not meant to exclude artificial or
synthetic mixtures of the saponin with other compounds. A number of
non-limiting examples of saponins and their methods of preparation
are provided below.
[0067] Saponins suitable for use in an individual are soluble in
aqueous solution and can be reconstituted from lyophilized or dried
saponins. Specific saponins useful for the present invention
include, but are not limited to, the "Quil-A" adjuvant preparation
sold by Superfos of Denmark, and the chromatographic fractions with
adjuvant activity that are described in U.S. Pat. Nos. 5,057,540
and 5,583,112, particularly fractions QS-21 (also referred to in
the patents as QA-21) and QS-7.
[0068] Also useful in the methods and compositions of the present
invention are chemically modified saponins that retain immune
stimulating activity. According to Kensil et al., U.S. Pat. No.
5,583,112, the contents of which are fully incorporated by
reference herein, the carboxyl group on the glucuronic acid of
saponins from Quillaja saponaria Molina can be conjugated to a
protein, a peptide, or a small molecule containing a primary amine.
According to Higuchi et al., 1987, Phytochemistry 26:229, saponins
from Quillaja saponaria may be deacylated by alkaline-catalyzed
hydrolysis. According to Marciani et al., U.S. Pat. No. 5,977,081,
the contents of which are fully incorporated by reference herein,
the carboxyl group on the glucuronic acid of nonacylated or
deacylated saponins from Quillaja saponaria may be conjugated to a
lipid, fatty acid, polyethylene glycol, or terpene.
[0069] Alternatively, an active fragment or synthetically modified
derivative of a fragment or a native saponin(s) may be utilized,
such as those described in Soltysik et al., 1995, Vaccine
13(15):1403-1410; Marciani et al., 2000, Vaccine 18:3141-3151. Such
modifications include but are not limited to removals or
substitutions of saccharide residues, addition of saccharide
residues, and removal, substitution and/or addition of acyl
chains.
[0070] The methods and compositions of the present invention may
also employ saponins isolated from plant species other than
Quillaja, such as Gypsophila or Saponaria officinalis (Bomford et
al, 1992, Vaccine 10(9):572-577, incorporated by reference herein
in its entirety), and Chenopodium quinoa saponins (Estrada et al.,
1998, Comp. Immunol. Microbiol. Infect. Dis. 21(3):225-36,
incorporated by reference herein in its entirety).
[0071] In certain embodiments of the invention, compositions of the
invention comprises saponins in combination with excipients.
Preferably, the saponin is QS-21 and the excipients are selected
from nonionic surfactants, polyvinyl pyrolidone, human serum
albumin, and various unmodified and derivatized cyclodextrins. More
preferably, in these embodiments, the nonionic surfactants are
selected from Polysorbate 20, Polysorbate-40, Polysorbate-60, and
Polysorbate-80. The polyvinyl pyrolidone may preferably be Plasdone
C15, a pharmaceutical grade of polyvinyl pyrolidone. Preferred
cyclodextrins are hydroxypropyl-.beta.-cyclodextrin,
hydroxypropyl-.gamma.-cyclodextrin, and methyl-.beta.-cyclodextrin.
Preferably, the cyclodextrins are .beta.-cyclodextrins. Examples of
the excipients include those described in PCT/US98/17940,
incorporated by reference herein in its entirety.
[0072] In another embodiment of the invention, compositions of the
invention comprises saponins in combination with immunostimulatory
polymers. Preferably, the saponin is QS-21 and the
immunostimulatory polymers are selected from cytokines, muramyl
dipeptides and tripeptide derivatives, CpG dinucleotides, CpG
oligonucleotides, monophosphoryl Lipid A, and polyphosphazenes.
Examples of the immunostimulatory polymers include those described
in PCT/US00/23688.
[0073] There are multiple acceptable techniques for extraction and
isolation of saponins from Quillaja saponaria Molina bark.
Acceptable procedures for purifying the saponins of the present
invention from Quillaja saponaria Molina bark, measuring the
saponins for immune adjuvant activity, and characterizing the
substantially pure saponins are disclosed in U.S. Pat. Nos.
5,057,540 and 5,583,112.
[0074] Aqueous extracts of Quillaja saponaria bark are also
available commercially. These are dark brown, foamy extracts that
contain many compounds (tannins, polyphenolics, saponins) that can
be analyzed by a method such as reversed phase BPLC.
[0075] An example of a reversed phase HPLC analysis of a typical
bark extract that is suitable for purification of saponins is shown
in FIG. 1 of U.S. Pat. No. 6,231,859, which is incorporated herein
in its entirety.
[0076] Partial purification to enrich the saponin fraction and to
remove the majority of tannins and polyphenolics can be
accomplished by dialysis of the extract against water through a
10,000 molecular weight membrane or ultrafiltration. The saponin
fraction is retained.
[0077] Alternatively, an aqueous saponin extract can be pretreated
with polyvinylpolypyrrolidone to remove high molecular weight
tannins and polyphenolics through absorption of these
compounds.
[0078] Residual tannins and polyphenolics can then be removed from
the saponin fraction by diafiltration against water. The saponin
fraction, which forms micelles, is retained by ultrafiltration
membranes of 10,000 to 30,000 molecular weight cutoff pore size.
This yields a partially purified extract that consists
predominantly of diverse saponins.
[0079] Separation of saponins can be accomplished by chromatography
in organic solvents or organic solvent/water mixtures. A separation
of saponins on silica was described in U.S. Pat. No. 5,057,540.
This yields saponins of intermediate purity (enriched in an
individual saponin, but less than substantially pure).
[0080] Alternatively, other solvent systems on silica gel or the
use of reverse phase chromatography can be used to accomplish the
initial separation of saponins. This initial purification step can
then typically be followed by reversed phase chromatography or
similar HPLC step to purify the saponins to near homogeneity.
[0081] For example, saponin extract may be recovered from plant
cell material freshly extracted from Quillaja trees. Dialyzed
extract is then purified on an ion exchange column, e.g., the DE-52
type, followed by Sephadex G50 gel filtration. Ultrafiltration may
be used instead of gel filtration. The purified saponin composition
is then subjected to RP-HPLC analysis on a VYDAC C4 column, eluted
with 30-45% acetonitrile in a 0.15% aqueous TFA-solution.
[0082] The substantially pure saponins useful in the present
invention may also be isolated from fresh plant material consisting
of substantially living cells as disclosed in WO 95/09179, or the
previously described procedures.
[0083] The same procedure may be performed on plant cell material
obtained by means of tissue culture or suspension cell culture.
See, e.g., U.S. Pat. No. 5,716,848, which is incorporated herein by
reference in its entirety.
[0084] General guidance on the use of saponins, Quil-A, and QS-21
can be found in the referenced patents. The amount of saponin
present in a pharmaceutically effective composition should contain
about 0.1 to 5,000 micrograms or more of a saponin. The amount of
saponin present in a pharmaceutically effective composition is more
preferably from about 1 to about 1000 micrograms, more preferably
from about 5 to 500 micrograms, and most preferably from about 10
to 100 micrograms. In certain specific embodiments, the amount of
saponin present in a pharmaceutical composition of the invention is
1, 2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110,
125 or 150 micrograms.
[0085] Formulations of pharmaceutical compositions comprising
saponins and procedures for their manufacture can be found in the
literature and in the U.S. patents incorporated by reference into
this Description. Saponin formulations referred to are provided
herein as nonlimiting examples. In U.S. Pat. No. 5,583,112 at
column 22, lines 11-17, a pharmaceutically effective composition
for intradermal administration was made by reconstituting
lyophilized "Quil A", a crude saponin mixture, into a phosphate
buffered saline (PBS) solution and mixed with a solution containing
10 micrograms of Bovine Serum Albumin (BSA) to achieve a final
volume of 200 microliters for intradermal injection. The effective
amount of "Quil A" was found to be approximately 30-77 micrograms
of "Quil A" by dry weight.
[0086] In U.S. Pat. No. 5,583,112 at column 23, lines 35-38, a
pharmaceutically effective composition for subcutaneous
administration was made by mixing a stock saline solution of
ovalbumin and 10 micrograms of QS-21.
[0087] Also in U.S. Pat. No. 5,583,112 at Column 23, lines 35-38, a
pharmaceutically effective composition for subcutaneous
administration was made by chemically crosslinking QS-21 to
lysozyme as described in Example 18 of the patent and resuspending
lyophilized QS-21/lysozyme conjugate into 200 microliters of PBS
(pH 7) for a final concentration of 10 micrograms of lysozyme and
1.6 micrograms of QS-21.
[0088] In a paper by Wu et al. (1994, Cellular Immunology
154:393-406), a pharmaceutically effective composition for
subcutaneous or intraperitoneal administration is disclosed
containing 25 micrograms ovalbumin absorbed to 250 micrograms of
Al(OH).sub.3 and 20 micrograms of QS-21 per immunization dose.
[0089] In another paper by Wu et al (1994, J. Immun.
148(5):1519-1525), a pharmaceutically effective composition for
immunization is disclosed containing 25 micrograms of a truncated
recombinant HIV-1 envelope protein absorbed to 250 micrograms of
Al(OH).sub.3 and 10 micrograms of QS-21 in a sterile saline per
immunization dose.
[0090] As a last non-limiting example, a pharmaceutically effective
vaccine has recently been tested in human patients containing 5-500
micrograms of a synthetic nonapeptide and 100 micrograms of QS-21
in 500 microliters PBS (pH 7.4) per intradermally-administered dose
(Lewis et al., 2000, Int. J. Cancer 87(3):391-398).
[0091] The optimum amount of a specific saponin for use with a
specific composition of the invention may vary. Optimization of the
specific saponin amount for a given composition is, as demonstrated
by the examples cited above, well within the purview of the skilled
artisan.
[0092] 4.3. Compositions of the Invention and Uses Thereof
[0093] The present invention encompasses therapies which involve
administering an immunoreactive reagent and a saponin to an animal,
preferably a mammal, and most preferably a human, for preventing,
treating, or ameliorating symptoms associated with a disease,
disorder, or infection. Prophylactic and therapeutic compounds of
the invention include, but are not limited to, immunoreactive
reagents and a saponin. Immunoreactive reagents such as antibodies
may be provided in pharmaceutically acceptable compositions as
known in the art or as described herein. The compositions of the
invention can also be used in conjunction with other forms of
therapy for a particular disease.
[0094] Compositions of the present invention can be administered to
an animal, preferably a mammal and most preferably a human, to
treat, prevent or ameliorate one or more symptoms associated with a
disease, disorder, or infection. In a preferred embodiment, the
composition of the invention exists outside of the body.
Preferably, the immunoreactive reagent of the invention has been
established to have some therapeutic benefit in the absence of
saponin, and recognizes an epitope on a cell or molecule associated
with the cause or symptoms of a disease, disorder or infection.
[0095] For example, the compositions and methods of the invention
can also be used to prevent, inhibit or reduce the growth or
metastasis of cancerous cells. In a specific embodiment, a
composition comprising an immunoreactive reagent, such as an
antibody, and saponin inhibits or reduces the growth or metastasis
of cancerous cells by at least 99%, at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
at least 50%, at least 45%, at least 40%, at least 45%, at least
35%, at least 30%, at least 25%, at least 20%, or at least 10%
relative to the growth or metastasis in absence of said
composition.
[0096] Each composition of the invention should contain at least
one immunoreactive reagent (as defined herein, e.g., an antibody)
and a saponin, and can then further comprise other reagents such as
cytokines, growth factors, immunostimulatory oligonucleotides, and
the like. One or more immunoreactive reagents that
immunospecifically bind to one or more target antigens may be used
locally or systemically in the body as a therapeutic. The
immunoreactive reagents may also be advantageously utilized in
combination with other such reagents such as monoclonal or chimeric
antibodies, or with immune active compounds such as lymphokines or
hematopoietic growth factors, such as, e.g., IL-2, IL-3 and IL-7 or
immune response modifiers (IRMs, 3M Pharmaceuticals, St. Paul,
Minn.), which, for example, serve to increase the number and/or
activity of immune effector cells which act in conjunction with the
immunoreactive reagent. In addition, immunostimulatory
oligonucleotides may be used in combination with the saponin and
immunoreactive reagents. Such oligonucleotides are known to enhance
the immune response. Woolridge, et al., 1997, Blood 89:2994-2998.
Such oligonucleotides are described in International Patent
Publication Nos. WO 01/22972, WO 01/51083, WO 98/40100 and WO
99/61056, each of which is incorporated herein in its entirety, as
well as U.S. Pat. Nos. 6,207,646 and 6,194,388, each of which is
incorporated herein in its entirety. Such immunostimulatory
oligonucleotides can comprise an unmethylated CpG motif. Other
kinds of immunostimulatory oligos such as phosphorothioate
oligodeoxynucleotides containing YpG- and CpR-motifs have been
described by Kandimalla et al. in "Effect of Chemical Modifications
of Cytosine and Guanine in a CpG-Motif of Oligonucleotides:
Structure-Immunostimulatory Activity Relationships." Bioorganic
& Medicinal Chemistry 9:807-813 (2001), incorporated herein by
reference in its entirety. Methods of determining the activity of
such oligonucleotides can be performed as described in the
aforementioned patents and publications. Moreover,
immunostimulatory oligonucleotides can be modified within the
phosphate backbone, sugar, nucleobase and internucleotide linkages
in order to modulate the activity. Such modifications are known to
those of skill in the art.
[0097] The immunoreactive reagents and saponins of this invention
may also be advantageously utilized in combination with one or more
drugs used to treat a disease, disorder, or infection such as, for
example anti-cancer agents, anti-inflammatory agents, or
anti-bacterial/fungal or anti-viral agents. Examples of anti-cancer
agents include, but are not limited to, cisplatin, ifosfamide,
paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-11,
topotecan, 9-ANTIGENIC COMPOSITION, and GG-211), gemcitabine,
vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin,
vinorelbine, temodal, and taxol.
[0098] In a specific embodiment, immunoreactive reagents
administered to an animal are of a species origin or species
reactivity that is the same species as that of the animal. Thus, in
a preferred embodiment, human or humanized antibodies are
administered to a human patient for therapy or prophylaxis.
[0099] The invention provides methods of treatment, prophylaxis,
and amelioration of one or more symptoms associated with a disease,
disorder or infection by administering to a subject of an effective
amount of an immunoreactive reagent and a saponin, or
pharmaceutical composition comprising an immunoreactive reagent and
a saponin. In a preferred aspect, the immunoreactive reagent and
saponin are substantially purified (i.e., substantially free from
substances that limit its effect or produce undesired
side-effects). In a specific embodiment, the subject is an animal,
preferably a mammal such as non-primate (e.g., cows, pigs, horses,
cats, dogs, rats etc.) and a primate (e.g., monkey such as a
cynomolgous monkey and a human). In a preferred embodiment, the
subject is a human.
[0100] Various delivery systems are known and can be used to
administer an immunoreactive reagent, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the immunoreactive reagent, etc. Methods of
administering an immunoreactive reagent and saponin or a
pharmaceutical composition comprising the same include, but are not
limited to, parenteral administration (e.g., intradermal,
intramuscular, intraperitoneal, intravenous and subcutaneous),
epidural, and mucosal (e.g., intranasal and oral routes).
Preferably, the immunoreactive reagent is administered
intravenously, while the saponin is not administered intravenously.
In a specific embodiment, immunoreactive reagents, for example,
antibodies, are administered intramuscularly, intravenously, or
subcutaneously. The compositions may be administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local. In addition, pulmonary administration can
also be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913;
5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO
97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which
is incorporated herein by reference in its entirety. In a preferred
embodiment, an immunoreactive reagent is administered using
Alkermes AIR.TM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.). Preferably, the saponin is not delivered
by pulmonary administration.
[0101] In accordance with the present invention, a composition of
the invention, comprising an immunoreactive reagent and a saponin
is administered to a human subject with cancer, an infectious
disease, or a neurodegenerative or amyloid diseases as a treatment.
In one embodiment, "treatment" or "treating" refers to an
amelioration of cancer, an infectious disease, or a
neurodegenerative or amyloid disease, or at least one discernible
symptom thereof. In another embodiment, "treatment" or "treating"
refers to an amelioration of at least one measurable physical
parameter associated with cancer, an infectious disease, a
neurodegenerative or amyloid disease, not necessarily-discernible
by the subject. In yet another embodiment, "treatment" or
"treating" refers to inhibiting the progression of a cancer, an
infectious disease, a neurodegenerative or amyloid disease, either
physically, e.g., stabilization of a discernible symptom,
physiologically, e.g., stabilization of a physical parameter, or
both. In yet another embodiment, "treatment" or "treating" refers
to delaying the onset of a cancer, a neurodegenerative or amyloid
disease.
[0102] In certain embodiments, the compositions of the present
invention are administered to a human subject as a preventative
measure against such cancer, an infectious disease, a
neurodegenerative or amyloid disease. As used herein, "prevention"
or "preventing" refers to a reduction of the risk of acquiring a
given cancer, infectious disease, neurodegenerative or amyloid
disease. In one mode of the embodiment, the compositions of the
present invention are administered as a preventative measure to a
human subject having a genetic predisposition to a cancer,
infectious disease, neurodegenerative or amyloid disease. In
another mode of the embodiment, the compositions of the present
invention are administered as a preventative measure to a subject
having a non-genetic predisposition to a cancer, or to a subject
facing exposure to an agent of an infectious disease.
[0103] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment or prevention. In one embodiment, the
treatment or prevention may be achieved by, for example, and not by
way of limitation, local infusion, by injection, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or
fibers. Preferably, care is taken to use materials to which the
immunoreactive reagent does not absorb. In a particular embodiment,
the immunoreactive reagent is administered systemically, for
example, by i.v., and the saponin is administered locally to the
area in need of treatment or prevention.
[0104] In another embodiment, the composition can be delivered in a
vesicle, in particular a liposome (see Langer, Science,
249:1527-1533, 1990; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3
17-327; see generally ibid.).
[0105] In yet another embodiment, the composition can be delivered
in a controlled release or sustained release system. Any technique
known to one of skill in the art can be used to produce sustained
release formulations comprising one or more antibodies, or one or
more fusion proteins. See, e.g., U.S. Pat. No. 4,526,938; PCT
publication WO 91/05548; PCT publication WO 96/20698; Ning et al.,
"Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy & Oncology,
39:179-189, 1996; Song et al., "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology, 50:372-397, 1995; Cleek et al., "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Intl. Symp. Control. Rel. Bioact. Mater.,
24:853-854, 1997; and Lam et al., "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759-760, 1997,
each of which is incorporated herein by reference in its entirety.
In one embodiment, a pump may be used in a controlled release
system (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.
Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; and Saudek et
al., 1989, N. Engl. J. Med. 321:574). In another embodiment,
polymeric materials can be used to achieve controlled release of
immunoreactive reagents (see e.g., Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.
23:61; see also Levy et al., 1985, Science 228:190; During et al.,
1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg.
71:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S.
Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No.
5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No.
WO 99/20253). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target (e.g.,
the lungs), thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0106] Other controlled release systems are discussed in the review
by Langer, 1990, Science 249:1527-1533).
[0107] The invention also provides that an immunoreactive reagent,
for example an antibody, is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
immunoreactive reagent. In one embodiment, the immunoreactive
reagent and saponin are supplied together or separately as dry
sterilized lyophilized powders or water free concentrates in one or
more hermetically sealed containers and can be reconstituted, e.g.,
with water or saline to the appropriate concentration for
administration to a subject. The effective dosage of each
immunoreactive reagent, and can be estimated initially from in
vitro assays. It also depends on the nature of the target antigen,
the density of the antigen in the tumors, the tumor type, the
manner of administration, which can be optimized by a person
skilled in the art without undue experimentation. Usual effective
dosages for injection range from about 0.1 to 5 mg/kg/day,
preferably from about 1 to 4 mg/kg/day, and more preferably from 2
to 4 mg/kg/week. Preferably, the immunoreactive reagent is supplied
as a dry sterile lyophilized powder in a hermetically sealed
container at a unit dosage of at least 5 mg, more preferably at
least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at
least 45 mg, at least 50 mg, or at least 75 mg.
[0108] In other embodiments, the saponin is QS-7, QS-17, QS-18,
QS-21, QS-21-V1, or QS-21-V2. Preferably, the saponin is supplied
at a unit dosage of at least 1 microgram, more preferably at least
10 micrograms, at least 15 micrograms, at least 25 micrograms, at
least 35 micrograms, at least 45 micrograms, at least 50
micrograms, or at least 75 micrograms. In a preferred embodiment,
the amount of saponin in the pharmaceutical composition is from
about 0.1 to about 1000 micrograms. In a preferred embodiment, the
amount of saponin is about 1, 2, 3, 5, 10, 15, 20, 25, 30, 40, 50,
60, 70, 80, 90, 100, 110, 125, 150, 200, 250, 300 or 500
micrograms. In a particularly preferred embodiment, the amount of
saponin is 100 micrograms. In a particularly preferred embodiment,
the saponin is QS-21. The amount of QS-21 in the pharmaceutical
compositions is preferably about 1 microgram or more. In a
particularly preferred embodiment, the amount of QS-21 is from
about 10 to about 1000 micrograms. In a particularly preferred
embodiment, the amount of QS-21 is about 10 to 100 micrograms, or
20 to 50 micrograms, preferably 25 or 50 micrograms. The
lyophilized immunoreactive reagent and saponin should be stored at
between 2 and 8.degree. C. in its original container and should be
administered within 12 hours, preferably within 6 hours, within 5
hours, within 3 hours, or within 1 hour after being reconstituted.
In an alternative embodiment, an immunoreactive reagent and saponin
are supplied in liquid form in a hermetically sealed container
indicating the quantity and concentration of the saponin and
immunoreactive reagent. Preferably, the liquid form of the
immunoreactive reagent is supplied in a hermetically sealed
container at least 1 mg/ml, more preferably at least 2.5 mg/ml, at
least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15
mg/ml, or at least 25 mg/ml. Preferably, the liquid form of the
saponin is supplied in a hermetically sealed container at least 1
mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at
least 8 mg/ml, at least 10 mg/ml, at least 15 mg/mil, or at least
25 mg/ml.
[0109] The present invention also provides pharmaceutical
compositions. Such compositions comprise a prophylactically or
therapeutically effective amount of an immunoreactive reagent and a
saponin, and a pharmaceutically acceptable carrier. In a specific
embodiment, the term "pharmaceutically acceptable" means approved
by a regulatory agency of the Federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" refers to a diluent, adjuvant (e.g., MPL,
immunostimulatory oligonucleotides, Freund's complete and
incomplete, mineral gels such as aluminum hydroxide, surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol,
and potentially useful adjuvants for humans such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum), excipient, or vehicle
with which the therapeutic is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents: These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a prophylactically or therapeutically effective amount of
the immunoreactive reagent and saponin, preferably in purified
form, together with a suitable amount of carrier so as to provide
the form for proper administration to the patient. The formulation
should suit the mode of administration.
[0110] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection.
Preferably, the saponin is not administered intravenously.
[0111] Generally, the ingredients of compositions of the invention
are supplied as a kit either separately or mixed together in unit
dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration. In another embodiment, a kit of the invention
further comprises a needle or syringe, preferably packaged in
sterile form, for injecting the composition, and/or a packaged
alcohol pad. Instructions are optionally included for
administration of the compositions of the invention by a clinician
or by the patient.
[0112] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0113] The amount of the composition of the invention which will be
effective in the treatment, prevention or amelioration of one or
more symptoms associated with a disease, disorder, or infection can
be determined by standard clinical techniques. The precise dose to
be employed in the formulation will depend on the route of
administration, the age of the subject, and the seriousness of the
disease, disorder, or infection, and should be decided according to
the judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model (e.g., the cotton rat or
Cynomolgous monkey) test systems. Models and methods for evaluation
of the effects of saponins and antibodies, or other immunoreactive
reagents are known in the art. (Wooldridge et al., Blood, 1997, 89
(8): 2994-2998, incorporated by reference herein in its
entirety).
[0114] For antibodies, the therapeutically or prophylactically
effective dosage administered to a subject is typically 0.1 mg/kg
to 200 mg/kg of the subject's body weight. Preferably, the dosage
administered to a subject is between 0.1 mg/kg and 20 mg/kg of the
subject's body weight and more preferably the dosage administered
to a subject is between 1 mg/kg to 10 mg/kg of the subject's body
weight. The dosage will, however, depend upon the extent to which
the serum half-life of the molecule has been increased. Generally,
human antibodies have longer half-lives within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of immunoreactive reagents may be
reduced also by enhancing uptake and tissue penetration (e.g., into
the lung) of the immunoreactive reagents such as, for example,
lipidation.
[0115] Treatment of a subject with a therapeutically or
prophylactically effective amount of an immunoreactive reagent and
saponin can include a single treatment or, preferably, can include
a series of treatments. In a preferred example, a subject is
treated with an immunoreactive reagent in the range of between
about 0.1 to 30 mg/kg body weight, one time per week for between
about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. In a preferred example, a subject is
treated with a saponin in the range of between about 0.1 to 30
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. In other embodiments, the pharmaceutical composition of the
invention is administered once a day, twice a day, or three times a
day. In other embodiments, the pharmaceutical composition is
administered once a week, twice a week, once every two weeks, once
a month, once every six weeks, once every two months, twice a year
or once per year. It will also be appreciated that the effective
dosage of the immunoreactive reagents used for treatment may
increase or decrease over the course of a particular treatment. The
immunoreactive reagent and the saponin can be administered
simultaneously. In a preferred embodiment, the saponin and
immunoreactive reagent are administered at different times. In one
embodiment, the immunoreactive reagent is administered prior to the
saponin, e.g., at least 7 days, 3 days, 2 days, 1 day, 16 hours, 12
hours, 8 hours, 4 hours, 2 hours or 1 hour prior. In a preferred
embodiment, the saponin is administered prior to the immunoreactive
reagent, e.g., at least 7 days, 3 days, 2 days, 1 day, 16 hours, 12
hours, 8 hours, 4 hours, 2 hours or 1 hour prior.
[0116] 4.4. Methods of Making Pharmaceutical Compositions
Comprising an Immunoreactive Reagent and a Saponin
[0117] The present invention encompasses methods of making
pharmaceutical compositions comprising a saponin and an
immunoreactive reagent.
[0118] In one embodiment, the saponin is combined with the
immunoreactive reagent. In a preferred embodiment, the saponin and
immunoreactive reagent are formulated separately, and are in
separate containers within a kit. Such a kit can optionally further
comprise instructions for the administration of the immunoreactive
reagent and saponin.
5. EXAMPLES
[0119] 5.1. Enhancement of Antibody Mediated Lysis In Vitro
[0120] Murine splenocytes (effector cells) are generated from the
spleens of naive 6-8 week old mice. These effector cells are
incubated with 1 to 10 ug/ml of QS-21 or appropriate amount of
another immunomodulatory saponin for 24 to 72 hours. At the end of
the incubation period, target cells (E.G7-OVA or MO4) are loaded
with 51Cr. Effector cells and labeled target cells are incubated at
defined effector:target ratios in the presence and absence of
monoclonal antibody to SIINFEKL/Class I MHC (1 to 10 ug/ml) at
4.degree. C. for 30 to 60 minutes. The lysis in the presence of
QS-21 and monoclonal is compared to controls without QS-21, without
monoclonal, or both. There is an enhanced lysis due to QS-21
(determined by the fold-enhancement of lysis due to
QS-21/monoclonal over monoclonal alone).
[0121] 5.2. Improvement of Protection in Tumor Challenge Model
[0122] C57B1/6 mice are inoculated by s.c. route in the flank with
M04 tumor (1.times.10.sup.5) or EG7-OVA tumor. At 24 to 48 hours
after inoculation, mice are injected by IP route with a monoclonal
antibody to SIINFEKL/Class I MHC, or by local SC route in the
presence or absence of QS-21 (10-20 ug). The antitumor effect of
the QS-21/monoclonal antibody treatment is compared to that of
monoclonal antibody treatment by monitoring the growth of the
tumors over a 30 to 60 day period (measurement with calipers).
Survival was determined, and significance with respect to time to
death was assessed using Cox regression analysis. Mice were also
observed daily for signs of toxicity including level of activity,
ruffled fur, diarrhea, and general appearance. A significant number
of mice treated with monoclonal antibody alone developed tumor
compared with those treated with antibody and QS-21. The benefit of
the QS-21 treatment is demonstrated by a delay in tumor
progression. Models and methods for evaluation of the effects of
saponins and antibodies, or other immunoreactive reagents are known
in the art. (Wooldridge et al., Blood, 1997, 89 (8): 2994-2998,
incorporated by reference herein in its entirety).
[0123] 5.3. Improved Opsonization of Bacteria by Use of OS-21
[0124] Improved opsonization of bacteria by saponin is demonstrated
in vitro by incubating effector cells for the opsonophagocytosis
assay (HL-60) with QS-21. The cells are evaluated for whether they
are more effective in opsonizing S. pneumonia or S. aureus at a
given antibody titer (for example a human serum sample with
opsonizing activity specific for S. pneumonia or S. aureus,
respectively).
[0125] 5.4. The Upregulation by QS-21 of Fc Receptors
[0126] Monocytes, natural killer cells, or polymorphonuclear cells
are incubated in the presence or absence of QS-21 (for example 1-10
ug/ml). The trypsinized cells are incubated at 4.degree. C. for 60
min with monoclonal antibodies specific to Fc a R, Fc gamma R1, Fc
gamma RII, or Fc gamma RIII. The cells are then incubated with an
anti-mouse IgG FITC probe, washed, fixed in parafomaldehyde, and
analyzed by FACScan. Upregulation of Fc receptors on these cells is
demonstrated.
[0127] In addition, upregulation of TNF-alpha, IL-6 and MIP-1-alpha
by QS-21 in macrophage cells was demonstrated. In bone marrow
derived dendritic cells, QS-21 was found to increase production of
MIP-1-alpha and IL-1, and to decrease the production of Il-12 and
MIP-1-beta.
[0128] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0129] Various publications are cited herein, the disclosures of
each of which are incorporated by reference herein in their
entireties.
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